I’m relatively new to CellProfiler, but familiar with the most basic functions and modules. One thing I’m struggling with, however, is to “define” the regions marked with WGA (plasma membrane marker, red channel) as regions of interest (ROI).
In my experiment, I have marked Cx43 (green), the nucleus (blue) and WGA (red), and I’m trying to quantify the signals from the green channel within defined ROI (i.e. the cytoplasm, plasma membrane). I tried to identify the WGA-stained parts as primary objects, but with this approach I get a lot of objects and thus huge output files, because each object gets a value. Is it possible to define the plasma membrane (WGA stained regions) as one single object?
Also, is my method for defining the cell and cytoplasm ROI good? I ask because my approach was more or less trial and error.
To define the “cells” ROI, in IdentifySecondaryObjects: I used the propagation method, WGA Outlines (from IdentifyPrimaryObjects) as an input image and the nuclei (from IdentifyPrimaryObjects) as input objects. Judging by the output it looked OK (see the attached image). Further, to identify the cytoplasm ROI, I substracted the nuclei from the “cells” in IdentifyTertiaryObject.
Thanks in advance. I’m really liking this software and hope to master it in the future.